Constitutive activation of mTOR signaling pathway in post-transplant lymphoproliferative disorders

Abstract

We examined activation of the mTOR signaling pathway in situ in the primary, normal reactive and patient-derived post-transplant lymphoproliferative disorder (PTLD) tissue samples. We accomplished this analysis by immunohistochemistry on formalin-fixed, paraffin-embedded specimens using a set of highly specific antibodies that permitted us to determine phosphorylation status of the key serines in the mTOR target proteins. Our results demonstrate that the mTOR signaling pathway is activated in reactive tissue in a highly distinct fashion with positive, typically enlarged cells being present primarily in the germinal center and, to a lesser degree, in interfollicular areas with mantle zone being conspicuously negative. We could demonstrate mTOR activation in the lesional cells in the entire spectrum of PTLD subtypes, regardless of their Epstein-Barr virus genome expression status. These data demonstrate the ubiquitous activation of the mTOR signaling pathway in PTLD and indicate that mTOR inhibitors may be effective in treatment and, notably, prevention of PTLDs given their immunosuppressive properties. Furthermore, our results define potential biomarkers of the therapeutic response. Because the constitutive mTOR activation has also been identified in cells isolated from other hematologic malignancies, the ability to examine the in vivo mTOR signaling may have implications reaching beyond the PTLD field.

Activation of mTORC1 Signaling Pathway in AIDS-Related Lymphomas

“…The prevalence of mTORC1 activation in various ARL histological types indirectly supported our previous notion 25 that the mobilization of the mTORC1 signaling pathway may be a feature of lymphocyte activation in general rather than specifically of the malignant cell transformation. Indeed, when we next evaluated eight cases of the reactive, HIV-associated lymphadenopathy, marked mTORC1 activation was identified in the germinal centers of the floridly hyperplastic follicles and, to a lesser degree, in enlarged lymphocytes in the interfollicular areas (Figure 4).…”

“…We used in this analysis the phospho-specific antibodies against S6rp (Ser235/236) and p-4E-BP1 (Ser65), which we have validated extensively in our recent studies. [23][24][25] Whereas these antibodies have proven highly suitable for immunohistochemistry, an antibody against phosphorylated p70S6K (Thr389) did not, due to its cross-reactivity with p-p90S6K, which is not phosphorylated by mTORC1. 25 The representative results of the current evaluation of the mTORC1 signaling pathway activation in ARL are depicted in Figures 1 to 3.…”

“…26,36 Our follow-up study demonstrated universal mTORC1 activation in the PTLD tissues, further supporting the notion of mTORC1-directed therapy in PTLD. 25 We, 26 and in a much greater depth other investigators, 27 have recently shown that cell lines derived primary effusion lymphoma, which invariably express genes of HHV8 and often also of EBV but in a restricted fashion, are also highly sensitive to mTORC1 inhibition both in vitro and in vivo, suggesting that the inhibition may also be effective in patients with primary effusion lymphoma. According to several independent studies on organ transplant patients who developed PTLD, substitution of the calcineurine inhibitor-based immunosuppressive therapy with an mTORC1 inhibitor resulted in a potent anti-PTLD effect leading to the full resolution of the PTLD lesions in some cases.…”

“…[23][24][25] In brief, 5-m sections from formalin-fixed, paraffin-embedded tissue specimens were deparaffinized in xylene and rehydrated in graded alcohol. After the deparaffinization, heat induced antigen retrieval was performed by boiling the slides in 10 mol/L citrate buffer for 20 minutes.…”

Potential therapeutic targets for chordoma: PI3K/AKT/TSC1/TSC2/mTOR pathway

“…p-p70S6K is generally used in preference to p-mTOR to assess the tumour response to mTOR antagonists and to predict which tumours may be responsive to such agents (for reviews see Boulay et al (2004); MacKenzie and von Mehren (2007)). Indeed, phosphorylated mTOR has been generally found to be expressed in relatively low numbers of tumours, and several studies make no reference to tumour mTOR expression when assessing the effect of rapamycin and or its analogues (Jaeschke et al, 2002;Gao et al, 2003;Krishnan et al, 2006;El-Salem et al, 2007;Lu et al, 2008; Although the TMA was built with 50 chordomas, the total number described for each antibody represents the number that was possible to analyse. The scoring system employed was as follows: negative in the absence of immunoreactivity, 'low' when the immunoreactivity was unequivocal but less strong than the positive control and 'high' when the immunoreactivity was at least as strong as the positive control.…”